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The Anti-inflammatory Effects of Ulinastatin
in Traumatic Patients with a Hemorrhagic Shock
Kyung Hye Park
The Graduate School
Yonsei University
Department of Medicine
The Anti-inflammatory Effects of Ulinastatin
in Traumatic Patients with a Hemorrhagic Shock
A Master's Thesis
Submitted to the Department of Medicine
and the Graduate School of Yonsei University
in partial fulfillment of the
requirement for the degree of
Master of Medical Science
Kyung Hye Park
January 2007
This certifies that the master’s thesis of
Kyung Hye Park is approved
___________________________
Thesis Supervisor : Kang Hyun Lee M.D.
___________________________
Thesis Committee Member : Sung Oh Hwang M.D.
___________________________
Thesis Committee Member : Jae Won Choi M.D.
The Graduate School
Yonsei University
January 2007
감사의감사의감사의감사의 글글글글
이 논문이 나오기까지 바쁘신 와중에도 아낌없는 지도를 해 주신 이강현
교수님께 가슴 속 깊이 감사드립니다. 또한 늘 성원해 주시고 지도해 주신
황성오 교수님, 귀중한 시간을 내어 조언해 주신 최재원 교수님께
감사드립니다. 그리고 관심을 가져주시고 조언해 주신 김현 교수님, 김선휴
선생님, 장용수 선생님께도 감사드립니다.
제 연구를 적극적으로 도와 준 응급의학과 의국원들과 응급실
간호사분들에게도 고마움을 전하고 싶습니다. 그리고 진단검사의학과 권오건
선생님과 퇴근 후에도 시간을 내어 혈청 검사를 해 주신 임상병리사 진혜경
선생님에게도 감사드립니다.
석사 논문을 완성시킨 기쁨을 누리게 해 주신 위의 모든 분들께
감사드립니다.
마지막으로 낳아주시고 길러주신, 물심양면으로 큰 딸을 응원해주신 아버지,
어머니께도 고개 숙여 감사드립니다. 교사와 공무원으로 바쁘게
생활하시면서도 석사학위를 따신 두 분의 노력이 제게 큰 힘이 되었습니다.
논문 쓰며 힘들어하는 나를 마음으로 위로해준 동생 경화, 경석이에게도
고마움을 전합니다.
이 논문을 내 사랑하는 가족들에게 바칩니다.
- i -
Index
Figure index · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · iii
Table index · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · iv
Abstract · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · v
I. Introduction · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 1
II. Subjects and Methods · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 2
1. Subjects · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 2
2. Methods · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 2
2.1. Patients · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 2
2.2. Administration of ulinastatin · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 3
2.3. Measurements of serum TNF-α, IL-6 and PMNE · · · · · · · · · · · · · 3
2.4. Analysis of results · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 4
3. Statistical analysis · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 6
III. Results · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 7
1. Demographic data for the patients · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 7
2. Comparison of laboratory data between the control group and the ulinastatin group · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 8
3. Comparison of serum TNF-α, IL-6 and PMNE levels between the control group and the ulinastatin group · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 10
3.1. Serum TNF-α levels · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 10
3.2. Serum IL-6 levels · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 11
3.3. Serum PMNE levels · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 13
4. Treatment and the final results · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 14
4.1. Transfusion, treatment and mortality · · · · · · · · · · · · · · · · · · · · · · · · · 14
4.2. Comparison of SIRS score, MODS score and APACHE III between
- ii -
the control group and the ulinastatin group · · · · · · · · · · · · · · · · · · · · · · · 16
IV. Discussion · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 19
V. Conclusion · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 24
References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 25
Abstract in Korean · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 30
- iii -
Figure Index
Fig. 1. The study design · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 5
Fig. 2. A. Effects of ulinastatin on serum TNF-α levels · · · · · · · · · · · · · · · · · 10
Fig. 2. B. Changes of serum TNF-α levels after admission · · · · · · · · · · · · · · · · · 11
Fig. 3. A. Effects of ulinastatin on serum IL-6 levels · · · · · · · · · · · · · · · · · · · · · 12
Fig. 3. B. Changes of serum IL-6 levels after admission · · · · · · · · · · · · · · · · · · · 12
Fig. 4. A. Effects of ulinastatin on serum PMNE levels · · · · · · · · · · · · · · · · 13
Fig. 4. B. Changes of serum PMNE levels after admission · · · · · · · · · · · · · · · · · 14
- iv -
Table Index
Table 1. Demographic data of the enrolled patients · · · · · · · · · · · · · · · · ·7
Table 2. Comparison of laboratory data between the control group and the ulinastatin group · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 8
Table 3. Transfusion, treatment modality and the final results between the control group and the ulinastatin group· · · · · · · · · · · · · · · · · · · · · ·15
Table 4. SIRS score, MODS score and APACHE III 48 hours after admission · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 17
Table 5. Comparison of SIRS score, MODS score and APACHE III between
the control group and the ulinastatin group · · · · · · · · · · · · · · · · · · · · 18
- v -
Abstract
The Anti-inflammatory Effects of Ulinastatin
in Traumatic Patients with a Hemorrhagic Shock
Kyung Hye Park
Dept. of Medicine
The Graduate School
Yonsei University
Background: Ulinastatin, a glycoprotein from human urine, inhibits the proteolytic
action and has an anti-inflammatory effect on tissues. Ulinastatin reduces the renal
dysfunction associated with the ischemia-reperfusion of the kidney as well as the blood
transfusion-induced Polymorphonuclear Leukocyte Elastase (PMNE) which may injure a
variety of tissues and organs. However, the effect of ulinastatin on traumatic hemorrhagic
shock has rarely been reported.
Purpose: The aim of this study was to investigate the use of ulinastatin in association
with the suppression of plasma proinflammatory cytokine and PMNE and the good
prognosis in the patients with traumatic hemorrhagic shock.
Subjects and Methods: Nineteen patients who were admitted to the emergency
department for trauma with hemorrhagic shock from June 2006 to October 2006 were
- vi -
enrolled. Eleven patients received ulinastatin at random. Ulinastatin 100,000 IU was
intravenously administered every 8 hours for a total of 300,000 IU. Measurements of
serum PMNE, Tumor Necrosis Factor Alpha (TNF-α) and Interleukin 6 (IL-6) were taken
before ulinastatin treatment, at 24 hours, 2 days, 3 days and 7 days after admission. We
compared the Systemic Inflammatory Response Syndrome (SIRS) score, the Multiple
Organ Dysfunction Syndrome (MODS) score and the Acute Physiology, Age, Chronic
Health Evaluation (APACHE) III between the control group and the ulinastatin group..
Results: There were no significant baseline differences between the control group and
the ulinastatin group. Furthermore, there were no significant differences in laboratory
data, treatment and mortality between the control group and the ulinastatin group. The
serum PMNE levels of the ulinastatin group were lower than the control at the second
hospitalized day (11.58±5.57 vs 4.33±1.21, p=0.19). Serum TNF-α and IL-6 levels of the
ulinastatin group decreased 24 hours after admission and were lower than the control,
however, there were no significant differences.
Conclusion: Ulinastatin 300,000 IU leads to decrease the serum PMNE in traumatic
patients with a hemorrhagic shock on the second day of hospitalization.
_______________________________________________________________________________
Key Words: Ulinastatin, Hemorrhagic shock, Polymorphonuclear Leukocyte Elastase,
Tumor Necrosis Factor Alpha, Interleukin 6
- 1 -
I. Introduction
Shock resulting from life-threatening hemorrhage induces the ischemic injuries of the all
organs as well as tissues. Furthermore, this kind of damage occurs during the
resuscitation period. In both hemorrhagic shock and systemic inflammations (i.e. burn,
acute pancreatitis, sepsis), neutrophils become active, inflammatory cytokine increases,
whereby these systemic metabolic change can lead to an acute respiratory distress
syndrome and microischemia of the liver, and can impair the function of the kidney, heart
and the brain. As a result, the multiple organ failure is the leading cause of mortality1-3).
Interleukin-6 (IL-6), Tumor Necrosis Factor Alpha (TNF-α) and Polymorphonuclear
Leukocyte Elastase (PMNE) begin to increase in the early inflammation stage,
stimulating various tissues and organs, and lead to a systemic inflammatory response. In
order to suppress this kind of cytokine, various studies regarding an antibody against
cytokine or a protease inhibitor have been attempted4).
Ulinastatin, a glycoprotein with a molecular weight of 67,000 daltons, derived from
human urine, has an anti-inflammatory activity to suppress PMNE, TNF-α, IL-6, IL-85-8).
In animal studies, the effects of ulinastatin on the artificially induced hemorrhagic shock
have been reported8, 9), whereas the studies about the inflammatory cytokine or PMNE
and clinical trials to hemorrhagic shock have not been reported. The purpose of this study
was to investigate the anti-inflammatory effects of ulinastatin on the proinflammatory
cytokines and PMNE in traumatic patients with a hemorrhagic shock.
- 2 -
II. Subjects and Methods
1. Subjects
This was a randomized controlled trial using nineteen adult patients who were admitted
to the emergency department for trauma from June to October 2006 were enrolled. The
enrolled patients with a traumatic hemorrhagic shock had arrived within six hours after an
accident and were 18 years or older.
Patients with any documented preexisting heart failure, chronic renal failure, liver
cirrhosis or chronic obstructive pulmonary disease were excluded. Other exclusion
criteria included cardiopulmonary resuscitation-performed patients, or patients with
severe brain injury which was the main cause of death or morbidity.
2. Methods
1) Patients
The enrolled patients were divided into two groups randomly. Patients who were
admitted on the even days composed the control group. Furthermore, those admitted on
the odd days made up the ulinastatin group to whom ulinastatin was administrated.
- 3 -
2) Administration of ulinastatin
Ulinastatin was administrated to the enrolled patients receiving blood transfusion and
fluid immediately after a hemorrhagic shock was diagnosed. Ulinastatin is commercially
known as Ulistin® (Ulinastatin 100,000 IU/2ml, Han Lim Pharm. Co., Ltd, Seoul, Korea).
100,000 IU ulinastatin with 100 ml normal saline for duration of 30 minutes at a time,
every 8 hours, for a total of three times.
3) Measurements of serum TNF-α, IL-6 and PMNE
In the ulinastatin group, measurements were taken of the plasma concentrations of
PMNE, IL-6 and TNF-α before injecting ulinastatin, 24 hours, 2, 3, and 7 days after
injection. In the control group, the plasma concentrations of PMNE, IL-6 and TNF-α
were measured upon admission to the Emergency Room (ER), 24 hours, 2, 3 and 7 days
after admission. After centrifuging the blood samples of the patients (MF 600, Hanil
Science Industrial, Seoul, Korea), the collected serum was kept in a freezer and then
dissolved to examine by use of Enzyme-Linked Immunosorbent Assay (ELISA; PhDTM
System, BIO RAD, USA).
Serum PMNE was measured in terms of the concentration of PMNE-α1-antitrypsin
complex using PMNE/α1-proteinase inhibitor complex ELISA kit (Calbiochem®, EMD
Biosciences, Inc., Darmstadt, Germany). Furthermore, serum IL-6 was measured using
Human IL-6 immunoassay kit (Quantikine®, R&D Systems, Inc., Minnesota, USA).
- 4 -
Lastly, serum TNF-α was measured using Human TNF-α /TNFSF1A immunoassay kit
(Quantikine®, R&D Systems, Inc., Minnesota, USA).
4) Analysis of results
Upon admission and 48 hours after admission to the ER, the Systemic Inflammatory
Response Syndrome (SIRS) score, the Multiple Organ Dysfunction Syndrome (MODS)
score, the Acute Physiology, Age, Chronic Health Evaluation (APACHE) III, transfusion
amounts, cause of death and the duration of ICU admission of the control group and the
ulinastatin group were compared (Fig. 1).
- 5 -
Fig. 1. The study design
a IRB: institutional review board
b GCS: Glascow coma scale
c CPR: cardiopulmonary resuscitation
d COPD: chronic obstructive pulmonary disease
e TNF: tumor necrosis factor
f IL: interleukin
g PMNE: polymorphonuclear leukocyte elastase
h SIRS: systemic inflammatory response syndrome
i MODS: multiple organ dysfunction syndrome
j APACHE: acute physiology, age, chronic health evaluation
- 6 -
3. Statistical analysis
Data were summarized and coded into a software (SPSS 13.0 for windows, SPSS Inc.,
Chicago, IL). Statistical analysis were performed with the Mann-Whitney U-test, Pearson
chi-square test for demographic data, comparing parameters of the two groups, and the
Wilcoxon test for comparison between the SIRS score, the MODS score and the
APACHE III within the same group. A p value <0.05 was considered statistically
significant.
- 7 -
III. Results
1. Demographic data for the patients
The control group consisted of eleven patients, and the ulinastatin group consisted of
eight. The mean age of each group were 48.0±17.1, 48.7±11.1 respectively, and of each
group, five and eight patients were males respectively. There was no significant
difference in the two groups (p>0.05). Regarding the injury mechanism, 8 were traffic
accidents, 3 motorcycle accidents, 5 falls, 1 stab injury, 1 cultivator accident and 1
collision by rock. Injury Severity Score (ISS) was 27.1±22.5, 20.6±11.6 respectively
(p=0.901), Revised Trauma Score (RTS) was 10.4±1.9, 10.3±1.8 respectively (p=0.858).
However, there was no significant difference (Table 1).
Table 1. Demographic data of the enrolled patients
Characteristics Control group (n=8) Ulinastatin group (n=11) P value
Age, years 48.0±17.1 48.7±11.1 1.000c
Sex, male/female, n 5/3 8/3 0.636d
Injury mechanism, n 0.040d
Pedestrian 2 1 Driver 0 1 Passenger 1 3 Motorcycle 3 0 Fall 0 5 Stab injury 0 1 etc 2 0
- 8 -
ISSa 27.1±22.5 20.6±11.6 0.901c
RTSb 10.4±1.9 10.3±1.8 0.858c * Mean±SD.
a ISS: injury severity score
b RTS: revised trauma score
c Mann-Whitney U-test
d Pearson Chi-square test
2. Comparison of laboratory data between the control group and the
ulinastatin group
White blood cell counts, neutrophil counts, hemoglobin, pH, base excess and lactate
were not significantly different between the control group and the ulinastatin group
(p>0.05) (Table 2).
Table 2. Comparison of laboratory data between the control group and the ulinastatin
group
Parameters Control group
(n=8) Ulinastatin group
(n=11) P valuea
At admission pH 7.42±0.72 7.35±0.15 0.265
Base excess (mmol/L) -6.1±3.4 -8.2±5.2 0.364
Lactate (mmol/L) 4.1±2.5 5.4±3.1 0.600
WBCb (109/L) 22.4±5.8 22.4±8.1 0.934
Neutrophil (109/L) 18.3±5.9 17.9±7.8 0.741
- 9 -
Hemoglobin (g/dL) 10.6±2.4 10.7±3.2 0.934
At 24 hours after admission pH 7.40±0.06 7.29±0.35 0.733
Base excess (mmol/L) -2.1±2.7 -4.7±14.5 0.435
Lactate (mmol/L) 2.6±2.6 3.7±3.3 0.425
WBC (109/L) 11.6±4.1 8.8±2.1 0.241
Neutrophil (109/L) 9.7±3.7 6.6±3.7 0.257
Hemoglobin (g/dL) 10.9±1.5 9.5±2.4 0.434
At 2 days after admission pH 7.42±0.03 7.44±0.06 0.699
Base excess (mmol/L) 0.1±1.5 0.9±3.4 0.606
Lactate (mmol/L) 0.7±0.2 1.4±0.7 0.134
WBC (109/L) 10.4±3.5 9.2±2.7 0.562
Neutrophil (109/L) 8.5±3.8 7.5±1.2 1.000
Hemoglobin (g/dL) 9.3±0.9 9.3±0.9 0.816
At 3 days after admission pH 7.42±0.03 7.41±0.04 0.698
Base excess (mmol/L) 0.2±0.8 -0.63±3.19 0.794
Lactate (mmol/L) 0.8±0.3 1.0±0.9 0.724
WBC (109/L) 8.2±2.9 8.3±1.6 0.563
Neutrophil (109/L) 6.5±3.1 6.8±1.5 0.465
Hemoglobin (g/dL) 9.4±1.1 9.1±0.9 0.353
At 7 days after admission pH 7.45±0.05 7.46±0.03 0.721
Base excess (mmol/L) 2.4±2.9 1.4±2.2 0.471
Lactate (mmol/L) 0.9±0.5 1.1±0.7 1.000
WBC (109/L) 10.2±3.5 9.8±5.4 0.655
Neutrophil (109/L) 7.4±3.6 7.7±3.1 0.732
Hemoglobin (g/dL) 10.5±1.1 10.3±1.4 0.654
* Mean±SD.
a Mann-Whitney U-Test
b WBC: white blood cell
- 10 -
3. Comparison of serum TNF-α, IL-6, and PMNE levels between the
control group and the ulinastatin group
1) Serum TNF-α levels
The serum TNF-α concentration increase up to the third day of hospitalization in the
control group, however, it was a lower concentration than the initial on the 7th day. On
the other hand, TNF-α levels decreased continuously within the ulinastatin group over the
period of 7 days. However, there were no significant difference between the mean of
serum TNF-α of the control group and the ulinastatin group (Fig. 2).
- 11 -
Fig. 2. A. Effects of ulinastatin on serum TNF-α levels. B. Changes of serum TNF-α
levels after admission. HD0 means before injection of ulinastatin.
(HD: hospitalized day)
2) Serum IL-6 levels
In the ulinastatin group, the serum concentration of IL-6 was 141.77±113.59 pg/ml after
1 day and then decreased to 52.82±29.68 pg/ml after the next day. But the control group
showed an increase of serum IL-6 from 78.31±52.95 pg/ml to 100.70±42.57 pg/ml by the
next day, however, decreased thereafter. The means of the control group and the
ulinastatin group were not different (Fig. 3).
- 12 -
Fig. 3. A. Effects of ulinastatin on serum IL-6 levels. B. Changes of serum IL-6 levels
after admission. HD0 means before infusion of ulinastatin. (HD : hospitalized
day)
- 13 -
3) Serum PMNE levels
The concentration of serum PMNE in the control group increased continuously and was
11.58±5.57 ng/ml on the second day of hospitalization. In the ulinastatin group, serum
PMNE kept up with the increased trend showing a small difference and lower average.
The plasma concentration of PMNE of the ulinastatin group on the second hospital day
was 4.33±1.21 ng/ml and was lower than that of the control group significantly (p=0.019).
The change between the second hospitalized day and the admitted day was statistically
significant (p=0.045) (Fig. 4).
- 14 -
Fig. 4. A. Effects of ulinastatin on serum PMNE levels. B. Changes of serum PMNE
levels after admission. HD0 means before infusion of ulinastatin. (HD :
hospitalized day)
4. Treatment and the final results
1) Transfusion, treatment and mortality
- 15 -
Within the 24 hours after admission to the ER, the total transfusion amount of packed
red blood cells, fresh frozen plasma, and platelet concentration were not different
between the two groups (p>0.05).
Within the control group, six of the eight patients underwent an operation, one had a
conservative treatment, and one moribund discharged. In the ulinastatin group, there were
six conservations and five operations. The treatment modalities in both groups were not
significantly different (p=0.117).
One patient died as a result of a hemorrhagic shock in the control group. In the
ulinastatin group, two patients died: one a hemorrhagic shock and the other of multiple
organ dysfunction syndrome (Table 3).
Table 3. Transfusion, treatment modality and the final results between the control group
and the ulinastatin group
Parameters Control group (n=8)
Ulinastatin group (n=11)
P value
In 24 hours transfusion pRBCa 5.0±5.1 9.3±9.8 0.376f
FFPb 0.8±1.5 2.9±3.3 0.089f
PCc 0 2.2±3.7 0.117f
Total transfusion pRBC 7.9±8.3 11.6±11.2 0.430f
FFP 3.8±9.1 3.4±3.4 0.137f
PC 0 2.9±4.0 0.062f
Treatment 0.117g
Conservation 1 6
- 16 -
Operation 6 5 Moribound discharge 1 0
ICUd admission 12.0±19.2 5.8±6.3 0.787f
Result 0.644g
Discharge alive 6 6 Transfer 1 3 Death 1 2
Cause of death 0.672g
Hypovolemia 1 1 MOFe 0 1
* Mean±SD.
a pRBC: packed red blood cells
b FFP: fresh frozen plasma
c PC: platelet concentrate
d ICU: intensive care unit
e MOF: multiple organ failure
f Mann-Whitney U-test
g Pearson Chi-square test
2) Comparison of SIRS score, MODS score and APACHE III between
the control group and the ulinastatin group
In the ulinastatin group, SIRS score and APACHE III decreased significantly (2.3±0.9
vs 0.8±0.9, p=0.03; 42.7±28.6 vs 24.9±23.8, p=0.02). MODS score decreased after 48
- 17 -
hours admission, but there was no difference in the two groups (4.0±3.7 vs 2.3±3.2,
p=0.10). In the control group, SIRS score and APACHE III decreased also (2.8±1.0 vs
0.6±0.8, p=0.03; 45.0±28.2 vs 16.9±13.1, p=0.02). However, the MODS score was not
different (2.3±1.5 vs 2.5±2.3, p=0.59) (Table 4).
The means of SIRS score, MODS score and APACHE III of two groups were not
different significantly (Table 5).
Table 4. SIRS score, MODS score and APACHE III 48 hours after admission
Parameters At study entry 48 hours after admission P valuea Control group (n=8)
SIRSb score 2.8±1.0 0.6±0.8 0.026
MODSc score 2.3±1.5 2.5±2.3 0.596
APACHEd III 45.0±28.2 16.9±13.1 0.018
Ulinastatin group (n=11) SIRS score 2.3±0.9 0.8±0.9 0.026
MODS score 4.0±3.7 2.3±3.2 0.102
APACHE III 42.7±28.6 24.9±23.8 0.017
* Mean±SD.
a Wilcoxon test.
b SIRS: systemic inflammatory response syndrome
c MODS: multiple organ dysfunction syndrome
d APACHE: Acute physiology, Age, Chronic health evaluation
- 18 -
Table 5. Comparison of SIRS score, MODS score and APACHE III between the control
group and the ulinastatin group
Parameters Control group
(n=8) Ulinastatin group
(n=11) P valuea
At admission
SIRSb score 2.8±1.0 2.3±0.9 0.224
MODSc score 2.3±1.5 4.0±3.7 0.530
APACHEd III 45.0±28.2 42.7±28.6 0.563
At 2 days after admission
SIRS score 0.6±0.8 0.8±0.9 0.702
MODS score 2.5±2.3 2.3±3.2 0.610
APACHE III 16.9±13.1 24.9±23.8 0.451
At 3 days after admission
SIRS score 0.3±0.5 0.3±0.5 1.000
MODS score 1.8±1.3 1.9±1.6 1.000
APACHE III 18.0±17.9 15.3±9.1 0.886
At 7 days after admission
SIRS score 0.4±0.8 1.0±1.0 0.250
MODS score 1.0±1.1 1.7±1.7 0.455
APACHE III 19.5±16.3 17.4±14.5 0.886
* Mean±SD.
a Mann-Whitney U-test.
b SIRS: systemic inflammatory response syndrome
c MODS: multiple organ dysfunction syndrome
d APACHE: Acute physiology, Age, Chronic health evaluation
- 19 -
IV. Discussion
One-hundred years ago, it was reported that human urine had the capacity to inhibit
trypsin10). In 1955, the protein with antitryptic activity in urine was isolated10). One main
function of bikunin, a urinary trypsin inhibitor, is to inhibit serine protease, especially
elastase and to suppress neutrophils, lymphocytes and macrophages increased by
infection and inflammation11). Now commonly known as ulinastatin, this urinary trypsin
inhibitor inhibits cell apoptosis by free radicals and lipid peroxidation in renal ischemia-
reperfusion injuries and has a suppressive effect against mitochondrial injury8).
It is known that the effect of ulinastatin is dose-dependant6, 12, 13). In this study, 300,000
IU, three times per day was used, for this is the commonly recommended dosage for acute
circulatory failure. This is comparison to the 50,000 IU/kg selected in canine
experiments9, 14), and 1,500,000 IU for a period of five days selected in clinical studies7, 15).
In Japan, Ulinastatin 6,000 IU/kg was permitted as the maximum for safety16). Although
side effects of ulinastatin are thought to include nausea, vomiting and hypersensitivity
reaction, etc, these side effects were not seen in this study, nor have they been seen in
others. Furthermore, in most animal studies, ulinastatin was administrated before the
induction of a hemorrhagic shock or a septic shock, and in clinical trials before a
laparotomy or blood transfusion5, 9, 14). However, ulinastatin was prescribed after
diagnosing a traumatic hemorrhagic shock in the ER in this study.
Serum TNF-α, IL-6, PMNE were chosen as the inflammatory mediators associated with
a hemorrhagic shock. Specifically, serum TNF-α is thought to be an important factor
- 20 -
among the three because it is secreted by the activated macrophage, stimulating other
inflammatory cytokines and bringing inflammatory cells to tissues17). Furthermore, IL-6
is secreted from the cells by early inflammatory reaction. In the rat model, trauma-
induced hemorrhagic shock increased plasma levels of the liver enzyme alanine
aminotransferase (ALT), a marker of liver injury, showing significant correlation with IL-
618). Witthaut et al.19) reported that serum IL-6 values in a septic shock were significantly
higher 150 times than those of the controls, therefore IL-6 was the main cytokine of
infection and inflammation. Finally, neutrophils secrete PMNE when inflammation
occurs, which can injure every tissue and organ20, 21).
Protease such as elastase is typically seen to be increased in case of inflammation and/or
infection, and any substance which can inhibit this protease results in an anti-
inflammatory effect22). α1-protease inhibitor (α1-PI) and ulinastatin are the intrinsic
physiologic protease inhibitor which can suppress PMNE activity. However, in
inflammatory tissues, α1-PI loses its ability to function in the acidic conditions, but
ulinastatin can continue to inhibit PMNE23). In addition, ulinastatin protects the
endothelial cell against neutrophil-mediated injury not only by inactivating the
extracellular elastase secreted by neutrophils, but also by acting directly on neutrophils
and suppressing the production and secretion of the activated elastase from them13).
There are the animal studies about the antibodies against rat IL-6 and TNF-α. Toth et
al.18) reported that ALT was suppressed by two thirds after injecting anti-IL-6 in a hepatic
injury of the resuscitated rat from a trauma-induced hemorrhagic shock. Furthermore,
Vallejo et al.24) found that treatment with TNF-α receptor antagonist abrogated
- 21 -
inflammatory mediators and left ventricular dysfunction before a hemorrhagic shock or at
the time of resuscitation.
Ulinastatin has effectiveness in animal studies with a septic shock and a hemorrhagic
shock induced experimentally9, 14). Specifically, in the septic shock canine model study,
ulinastatin improved blood pressure and lactic acid levels. Interestingly, although
ulinastatin does not have anti-microbial activity, the ulinastatin-treated group was found
to have a bacterial count that was significantly decreased, and a high survival rate14). It is
thought that ulinastatin might activate the reticuloendothelial system and the
phagocytosis14). Furthermore, in hemorrhagic shock, the protective effect of ulinastatin
might be associated with the up-regulation of Bcl-2, a kind of inhibitor of the cell
apoptosis8).
Based on the literature, we hypothesized that ulinastatin would inhibit the inflammatory
cytokines such as TNF-α, IL-6 and PMNE. But in actuality, there were no difference
found among the averages of TNF-α, IL-6 and PMNE concentrations except for PMNE
on the second day of hospitalization.
Aosasa et al.6) reported that ulinastatin decreased the TNF-α production of
lipopolysaccharide (LPS)-stimulated monocytes, but there was no significant difference.
Serum TNF-α concentration was low when ulinastatin was used before LPS stimulation
and the serum concentration of TNF-α was in inverse proportion to the amount of
ulinastatin. In this study, after ulinastatin was injected to the ulinastatin group, serum
TNF-α level was lower than the initial serum TNF-α levels. Nonetheless, serum TNF-α
concentrations of the control group increased until the third hospitalized day.
- 22 -
Serum IL-6 concentrations of the ulinastatin group decreased by half after one day of
ulinastatin injection and from the second day of hospitalization. Serum IL-6 concentration
was lower than initial IL-6 cincentration in the ER within the two groups. Nishiyama et
al.5) proposed that ulinastatin might be useful to inhibit blood transfusion-induced
increase of serum PMNE concentrations but not IL-6 after a laparotomy. In their study,
serum PMNE levels increased to a lesser extent than the control group by 50 percent.
Tani et al.15) used a total of 1,500,000 IU of ulinastatin for 5 days after laparotomy.
Within the control group and the urinary trypsin inhibitor (UTI) group, PMNE
concentrations were not different statistically but moved within a narrow range in the UTI
group. In another study, laparotomy was taken and ulinastatin was administered to the
patients at the same time. In this instance, although serum PMNE levels did not decrease
significantly, coagulation and fibrinolysis was inhibited significantly16).
The reference range of serum PMNE levels was reported as 20 180 ∼ ㎍/L25) or
21 165 ∼ ㎍/L5). We did not check the normal serum concentration of PMNE, but the
reference range was 0.15 3.0 ng/ml. The lowest value was 1.49 ng/ml and the highest ∼
value was 19.88 ng/ml. In the clinical study about laparotomy or blood transfusion, the
peak value of serum PMNE was seen immediately after an operation or a blood
transfusion. Ulinastatin administration were seen to gradually decrease serum
concentrations of PMNE, but the serum concentration of PMNE were shown to increase
three-fold in the control group5, 16). In this study, serum PMNE levels increased more than
two-fold on the second hospitalized day within the control group, but serum PMNE
concentration was suppressed from increasing in the ulinastatin group. In addition, serum
- 23 -
IL-6 showed its peak value upon admission to the ER, and then was found to gradually
decrease in the ulinastatin group, however in the control group, serum IL-6 levels after
one day of hospitalization was at its highest value, and then was decreased. Similarly, it
has been shown in other studies that within septic shock patients, serum IL-6
concentration was peaked on the first day of diagnosis and then decreased slowly, and
serum IL-6 concentration showed the maximum after 24 hours in the rat model
hemorrahgic shock study18, 19).
The laboratory data, physiologic results like the SIRS score, MODS score and APACHE
III and the mortality had no difference between the two groups. First of all, this may be
due to the small number of patients and that is one limitation of this study. Secondly, it
may be because ultimately the control group underwent the same medical procedures as
the ulinastatin group, such as a blood transfusion and fluid therapy, and having been
required an operation. Therefore, a larger number of patients and a longer period for
clinical study is needed. Furthermore, studies regarding the various dosages of ulinastatin
for a traumatic hemorrhagic shock patients are needed.
- 24 -
V. Conclusion
Ulinastatin was shown to prevent the increase of serum PMNE levels in traumatic
patients with a hemorrhagic shock.
- 25 -
References
1. Brun-Buisson C. The epidemiology of the systemic inflammatory response. Intensive
Care Med 2000;26:S64-74.
2. Maitra SR, Gestring M, El-Maghrabi MR. Alternations in hepatic 6-phosphofructo-2-
kinase/fructose-2,6-bisphosphatase and glucose-6-phosphatase gene expression after
hemorrhagic hypotension and resuscitation. Shock 1997;8:385-8.
3. Lee CC, Marill KA, CarterWA, Crupi RS. A current concept of trauma-induced
multiorgan failure. Ann Emerg Med 2001;38:170-6.
4. Riedemann NC, Neff TA, Guo RF, Bernacki KD, Laudes IJ, Sarma JV, et al.
Protective effects of IL-6 blockade in sepsis are linked to reduced c5a receptor
expression. J Immunol 2003;170:503-7.
5. Nishiyama T, Hanaoka K. Do the effects of a protease inhibitor, ulinastatin, on elastase
release by blood transfusion depend on interleukin 6? Crit Care Med 2001;29:2106–10.
6. Aosasa S, Ono S, Mochizuki H, Tsujimoto H, Ueno C, Matsumoto A. Mechanism of
the inhibitory effect of protease inhibitor on tumor necrosis factor α production of
monocytes. Shock 2001;15:101–5.
- 26 -
7. Sato Y, Ishikawa S, Otaki A, Takahashi T, Hasegawa Y, Suzuki M, et al. Induction of
acute-phase reactive substances during open-heart surgery and efficacy of ulinastatin.
Inhibiting cytokines and postoperative organ injury. Jpn J Thorac Cardiovasc Surg
2000;48:428–34.
8. Chen CC, Liu ZM, Wang HH, He W, Wang Y, Wu WD. Effect of ulinastatin on renal
ischemia-reperfusion injury in rats. Acta Pharmacol Sin 2004;25:1334-40.
9. Ohnishi H, Suzuki K, Niho T, Ito C, Yamaguchi K. Protective effects of urinary
trypsin inhibitor in experimental shock. Jpn J Pharmacol. 1985;39:137-44.
10. Fries E, Blom AM. Bikunin-not just a plasma proteinase inhibitor. Int J Biochem Cell
Biol 2000;32:125-37.
11. Pugia MJ, Lott JA. Pathophysiology and diagnostic value of urinary trypsin inhibitors.
Clin Chem Lab Med 2005;43:1-16.
12. Yano T, Anraku S, Nakayama R, Ushijima K. Neuroprotective effect of urinary
trypsin inhibitor against focal cerebral ischemia-reperfusion injury in rats.
Anesthesiology 2003;98:465-73.
- 27 -
13. Nakatani K, Takeshita S, Tsujimoto H, Kawamura Y, Sekine I. Inhibitory effect of
serine protease inhibitors on neutrophil-mediated endothelial cell injury. J Leukoc Biol
2001;69:241-7.
14. Tani T, Aoki H, Yoshioka T, Lin KJ, Kodama M. Treatment of septic shock with a
protease inhibitor in a canine model: a prospective, randomized, controlled trial. Crit
Care Med 1993;21:925-30.
15. Tani T, Abe H, Endo H, Hanasawa K, Kodama M. Effects of a urinary trypsin
inhibitor on acute circulatory insufficiency after surgical operation. Am J Surg
1998;175:142-5.
16. Nishiyama T, Yokoyama T, Yamashita K. Effects of a protease inhibitor, ulinastatin,
on coagulation and fibrinolysis in abdominal surgery. J Anesth 2006;20:179-82.
17. Cohen J. The immunopathogenesis of sepsis. Nature 2002;420:885-91.
18. Toth B, Yokoyama Y, Schwacha MG, George RL, Rue III LW, Bland KI, et al,
Insights into the role of interleukin-6 in the induction of hepatic injury after trauma-
hemorrhagic shock. J Appl Physiol 2004;97:2184-9.
- 28 -
19. Witthaut R, Busch C, Fraunberger P, Walli A, Seidel D, Pilz G, et al. Plasma atrial
natriuretic peptide and brain natriuretic peptide are increased in septic shock: impact of
interleukin-6 and sepsis associated left ventricular dysfunction. Intensive Care Med
2003;29:1696-1702.
20. Duswald KH, Jochum M, Schramm W, Fritz H. Released granulocytic elastase: An
indicator of pathobiochemical alterations in septicemia after abdominal surgery. Surgery
1985;98:892-9.
21. Pacholok SG, Davies P, Dorn C, Finke P, Hanlon WA, Mumford RA, et al.
Formation of polymorphonuclear leukocyte elastase: alpha 1 proteinase inhibitor
complex and A alpha (1-21) fibrinopeptide in human blood stimulated with the calcium
ionophore A23187. A model to characterize inhibitors of polymorphonuclear leukocyte
elastase. Biochem Pharmacol 1995;49:1513-20.
22. Mania-Pramanik J, Potdar SS, Vadigoppula A, Sawant S. Elastase:a predictive marker
of inflammation and/or infection. J Clin Lab Anal 2004;18:153-8.
23. Ogawa M, Nishibe S, Mori T, Neumann S. Effect of human urinary trypsin inhibitor
on granulocyte elastase activity. Res Commun Chem Pathol Pharmacol 1987;55:271-4.
- 29 -
24. Vallejo JG, Nemoto S, Ishiyama M, Yu B, Knuefermann P, Diwan A, et al.
Functional significance of inflammatory mediators in a murine model of resuscitated
hemorrhagic shock. Am J Physiol Heart Circ Physiol 2005;288:1272-7.
25. Neumann S, Gunzer G, Hennrich N, Lang H. "PMN-elastase assay": enzyme
immunoassay for human polymorphonuclear elastase complexed with alpha 1-
proteinase inhibitor. J Clin Chem Clin Biochem 1984;22:693-7.
- 30 -
Abstract in Korean
출혈성출혈성출혈성출혈성 쇼크가쇼크가쇼크가쇼크가 동반된동반된동반된동반된 외상외상외상외상 환자에서환자에서환자에서환자에서
UlinastatinUlinastatinUlinastatinUlinastatin 의의의의 항염증항염증항염증항염증 효과효과효과효과
연세대학교 대학원
의학과
박경혜
배경배경배경배경 및및및및 목적목적목적목적:::: Ulinastatin 은 사람의 소변에서 분리 정제된 당단백질로서 단백
분해 효소를 저해하고 항염증 작용이 있다. 또한 신장의 허혈 손상을 줄이고 수혈 후
주요 장기에 손상을 유발하는 Polymorphonuclear Leukocyte Elastase (PMNE)를
억제하는 효과가 있다고 알려져 있다. 그러나 외상에 의한 출혈성 쇼크에서는
ulinastatin 의 효과는 잘 알려져 있지 않다. 본 연구에서는 출혈성 쇼크를 동반한
외상 환자에서 ulinastatin 투여가 환자의 혈청 전염증 시토카인 (cytokine)과
PMNE의 발현을 억제하여 환자의 예후에 좋은 영향을 주는지 알아보고자 한다.
대상대상대상대상 및및및및 방법방법방법방법:::: 2006월 6월부터 10월까지 응급실에 내원한 출혈성 쇼크를
동반한 외상 환자를 대상으로 하였다. 응급실 내원 당시에 출혈성 쇼크가 진단된
환자 중 무작위로 정하여 실험군에 해당하는 환자에게 ulinastatin을 1회에 10만
- 31 -
단위 씩 8시간 간격으로 총 3회 투여하였다. 투여 전, 투여 후 24시간, 2일 째, 3일
째, 7일 째에 PMNE, Tumor Necrosis Factor Alpha (TNF-α), Interleukin 6 (IL-
6)를 측정하였고, 내원 당시와 내원 후의 Systemic Inflammatory Response
Syndrome (SIRS) score, Multiple Organ Dysfunction Syndrome (MODS) score와
Acute Physiology, Age, Chronic Health Evaluation (APACHE) III를 비교하였다.
결과결과결과결과:::: 대상 환자는 모두 19명으로 대조군은 8명, 실험군은 11명이었으며, 두 군의
Injury Severity Scale은 차이가 없었다 (p=0.091). 그리고 두 군의 혈액학적 검사
소견, 치료, 사망률 등에도 유의한 차이가 없었다. PMNE의 농도는 내원 2일 째
대조군이 11.58±5.57 ng/ml, 실험군이 4.33±1.21 ng/ml로 두 군 간의 유의한
차이를 보였다 (p=0.19). TNF-α, IL-6는 내원 1일 째부터 감소하면서 대조군보다
낮은 값을 보였으나 의미 있는 차이는 없었다.
결론결론결론결론:::: Ulinastatin 30만 단위는 출혈성 쇼크를 동반한 외상 환자의 혈청 PMNE
농도를 내원 2일 째에 ulinastatin을 투여하지 않은 환자보다 의미 있게 낮춘다
_______________________________________________________________________________
핵심되는핵심되는핵심되는핵심되는 말말말말:::: Ulinastatin, Hemorrhagic shock, Polymorphonuclear Leukocyte
Elastase, Tumor Necrosis Factor Alpha, Interleukin 6
Recommended